CN108653308B - Application of rosaceous flowering glycoside in treating Alzheimer disease - Google Patents
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Abstract
The invention discloses application of rosaceous cherry glycoside in treating Alzheimer disease. Experiments prove that the primrose glycoside can promote Abeta25‑35Proliferation rate and activity of damaged PC-12 cells, and inhibition of A beta25‑35The invention provides a new method for treating Alzheimer disease by apoptosis of damaged PC-12 cells.
Description
Technical Field
The invention belongs to the field of biomedicine, and relates to application of sakura glycosides in treating Alzheimer disease.
Background
Alzheimer's Disease (AD), also known as senile dementia, is a neurodegenerative Disease that occurs in the elderly and is characterized mainly by chronic irreversible memory loss and cognitive function obstruction, and it generally takes ten years from diagnosis to death, and it seriously harms people's health and brings countless emotional and economic burdens to families and society. With the improvement of the living standard of people in China, the average life of the population is prolonged, the aging becomes an important problem facing China at present, and AD becomes a serious problem influencing the living quality and living quality of people. There is currently no effective cure for AD and no successful case has been found in over 100 human trials for AD treatment over the last decade. These unprecedented challenges highlight the importance and necessity of finding new drugs to prevent and early interfere with AD (ash mullard. sting of Alzheimer's failures offset by up-coming preservation variants J. Nature reviews Drug discovery.2012,11: 657-.
More studies have found that apoptosis plays an important role in the development and progression of AD. Apoptosis, also known as programmed cell death, can be divided into five steps of apoptosis activation signaling, initiation, commitment, execution and clearance. Normal apoptosis is a process that is actively initiated to better maintain homeostasis (Sujoy Bhattacharya, Ramesh M.ray, Leonard R.Johnson. cyclin-dependent kinases regulating apoptosis of intracellular epithelial cells [ J ]. Apoptosis.2014,19(3):451-466), which is likely to cause neurodegenerative disease when neurons are stimulated by various harmful substances and once normal apoptosis is over-activated. Studies have shown that A.beta.may trigger neuronal apoptosis (E.J.Mufson, L.Mahady, D.Waters, et al.Hippocpal plastics degradation of Alzheimer's disease [ J ]. neuroscience.2015,309: 51-67).
There is evidence that endogenous estrogen consumption in postmenopausal women is one of the most important risk factors for the onset of AD. Estrogens can exert protective effects in the presence of various neurotoxicity conditions, such as glutamate excess, serum deprivation, and A β toxicity. Various examples in different neuronal types have demonstrated that estrogens can exert pleiotropic effects through both genomic and non-genomic mechanisms to prevent the development of a β toxicity. However, estrogen may predispose women to breast and uterine cancer, which undoubtedly jeopardizes the clinical use of estrogen. During the last years, phytoestrogens have gained widespread attention as potential replacements for estrogen therapy, and are recommended as potential new drug candidates for AD. The research on the correlation between the plant hormone and the Alzheimer disease has important significance for the clinical treatment of the AD.
Disclosure of Invention
In order to solve the problems in the prior art, one of the objects of the present invention is to provide a pharmaceutical composition for treating alzheimer's disease, which comprises the plant hormone sakura glycoside.
The other object of the present invention is to provide a method for protecting nerve cells, which is the administration of sakuranetin.
The invention also aims to provide the application of the sakuranetin in preparing the medicines for protecting nerve cells and treating diseases caused by damage of the nerve cells.
Specifically, the invention adopts the following technical scheme:
the invention provides a pharmaceutical composition for treating Alzheimer disease, which comprises an effective amount of primrose-hip glycoside.
Further, the pharmaceutical composition also comprises one or more of luteolin-7-O-beta-D neohesperidin, naringin, sakuranetin-7-O-beta-D-glucoside, luteolin-7-O-beta-D-glucoside, neoeriocitrin, eriodictyol and eriodictyol.
Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier or auxiliary material.
Further, the pharmaceutical composition is in the form of a powder, gel, emulsion or liquid.
Further, the pharmaceutical composition is in the form of a tablet, a wafer capsule, a gel capsule, a stick, a sachet, a vial, a dropper, or an injectable form.
The invention provides a method for protecting nerve cells, which is to administer an effective amount of primrose cherry blossom glycoside. Further, the method for protecting nerve cells comprises promoting proliferation of nerve cells or inhibiting apoptosis of nerve cells. Wherein the method is a method for non-diagnostic purposes.
Further, the nerve cell is a PC-12 cell.
Preferably, when the PC12 cells are injured PC-12 cells, the concentration of sakuranetin is 4 × 10-1μmol/L。
The invention provides application of rosaceous cherry glycoside in preparation of a medicament for protecting nerve cells.
The invention provides application of rosa laevigata glycoside in preparing a medicament for treating diseases caused by damage of nerve cells.
In the present invention, the pharmaceutically acceptable carrier is one or more of a physiologically acceptable solvent, dispersion medium, integument, antibacterial and antifungal agent, isotonic or absorption delaying agent, and the like, including any and all. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, or ethanol, and the like, and combinations thereof. In many cases, it will be desirable to include isotonic agents, for example, one or more of sugars, polyalcohols such as mannitol, sorbitol, sodium chloride and the like in the composition. The pharmaceutically acceptable carrier may also contain minor amounts of auxiliary substances such as one or more of wetting or emulsifying agents, preservatives or buffers, and the like, which enhance the effective duration or efficacy of rosaceous flowering glycosides and other plant hormones.
In a specific classification, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the field of medicine, and comprises excipients, such as one or more of starch, water and the like; one or more of a lubricant, such as glycerin or magnesium stearate, and the like; disintegrating agents, such as one or more of microcrystalline cellulose, agar, calcium carbonate or sodium bicarbonate; fillers, such as one or more of starch or lactose; a binder such as one or more of pregelatinized starch, dextrin, cellulose derivatives, alginate, gelatin, or polyvinylpyrrolidone, etc.; osmotic pressure regulators, such as one or more of glucose, sucrose, sorbitol, or mannitol; diluents such as water and the like; absorption accelerators such as quaternary ammonium compounds and the like; surfactants such as cetyl alcohol and the like; an adsorption carrier, such as one or more of kaolin, bentonite, etc.; lubricants, such as one or more of talc, calcium stearate, magnesium stearate, or polyethylene glycol; in addition, other adjuvants such as one or more of flavoring agents or sweeteners may also be added to the composition.
For example, an injectable preparation can be prepared by dissolving, suspending or emulsifying a plant hormone such as acteoside as an active ingredient in a suitable aqueous solvent (e.g., one or more of distilled water, physiological saline, or a solution of green) or oily solvent (e.g., one or more of vegetable oil such as olive oil, sesame oil, cottonseed oil, corn oil, or propylene glycol), wherein the solvent may contain a dispersing agent (e.g., one or more of polysorbate 80, polyoxyethylene hardened castor oil 60, polyethylene glycol, benzyl alcohol, chlorobutanol, or phenol), an osmotic pressure regulator (e.g., one or more of sodium chloride, glycerol, D9-mannose, D-sorbitol, or glucose, etc.). In this case, additives such as solubilizing agents (e.g., one or more of sodium salicylate, sodium acetate, or the like), stabilizers (e.g., human serum albumin, or the like), analgesics (e.g., benzyl alcohol, or the like), and the like may be added, if necessary.
Pharmaceutical compositions of rosaponin and other phytohormones can be formulated into various dosage forms using conventional manufacturing methods well known in the art, for example, by mixing the active ingredient with one or more carriers and then formulating into the desired dosage form. The dosage form comprises one or more of tablets, capsules, granules, suspensions, emulsions, solutions, syrups or injections, and the like, and one or more administration routes of oral administration or injection (including one or more of intravenous injection, intravenous drip, intramuscular injection or subcutaneous injection, and the like), mucosal dialysis and the like are adopted for treating or scientifically researching the AD and related diseases thereof.
For oral administration, it may be formulated into one or more conventional solid preparations such as tablets, powders, granules or capsules. In practice, the present invention of rosaceous flowering glycosides and derivatives thereof may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The sakuranetin and other plant extracts (if desired) can also be encapsulated in hard or soft shell gelatin capsules, compressed into tablets or added directly to the subject's diet. For oral therapeutic administration, the sakuranetin and other plant extracts may be added with excipients and used in the form of one or more of edible tablets, buccal tablets, lozenges, capsules, suspensions, syrups, wafers, or the like.
In order to administer the pharmaceutical composition of rosaniline of the present invention in addition to parenteral administration, it may be necessary to coat or co-administer rosaniline with a material that prevents its inactivation. Supplementary active compounds may also be added to the composition. In particular implementations, the rosaponin of the present invention is co-formulated and/or co-administered with one or more other therapeutic agents that may be used to treat a disease. Such a combination may advantageously utilize lower doses of therapeutic agents, thus avoiding potential toxicity or complications associated with each monotherapy.
Making into liquid preparation such as aqueous solution, oil suspension or other liquid preparation, such as syrup or elixir; for parenteral administration, it may be formulated into one or more of solution for injection, aqueous solution, oily suspension, etc.
Among the above-mentioned forms of use, preferred forms are one or more of tablets, coated tablets, capsules, suppositories, injections and the like, more preferred are one or more of tablets, capsules, injections and the like, and particularly preferred are injections.
Pharmaceutical compositions of rosaponin must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injectable solutions are prepared by incorporating the desired amount of the rosa laevigata glycoside in the appropriate solvent with one or a combination of the desired ingredients enumerated above, as required, followed by sterile filtration. Generally, dispersions are prepared by adding the rosaceous flowering plants to a sterile vehicle containing a basic dispersion medium and the required other ingredients as described above. In the case of sterile powders for the preparation of sterile injectable solutions, the recommended methods of preparation are vacuum drying and freeze drying. Proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent which delays absorption, for example, monostearate salts or gelatin.
The term "effective amount" means an amount sufficient to treat the disease at a reasonable benefit/risk ratio applicable to any medical treatment. The effective dosage level of the composition may be determined according to the type of the subject, the severity of the disease, the age and sex of the subject, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the excretion rate, the treatment time, the drug to be used in combination with the composition, and other known factors in the medical field. The pharmaceutical compositions of the present invention may be used alone or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. The compositions may be administered in one or more dosage forms. In view of all the above factors, it is important to administer the composition at the minimum amount capable of exhibiting the maximum effect without causing side effects, which can be readily determined by one skilled in the art.
The invention has the advantages and beneficial effects that:
the invention develops new medical application of the rosa laevigata glycoside and provides a new medicine source for preventing and treating the Alzheimer disease.
The rosa laevigata glycoside has wide sources, simple preparation process and wide application range, can play a role to the maximum extent and is easy to apply to clinic.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 detection of estradiol (E) by MTT method2) For Abeta25-35Effect on the viability of PC-12 cells
1. Cell culture
Rat adrenal pheochromocytoma cell PC-12 cell line (BIOSYNTHESIS BIOTECHNOLOGY CO., LTD) in DMEM medium containing 10% FBS and 1% P/S at 37 deg.C and 5% CO2Culturing in the incubator, changing the culture solution for 1 time in 2-3 days, performing conventional digestion and passage by using 0.25% of trypsin containing EDTA, and collecting the fine particles in logarithmic growth phaseCells were used for the experiments.
2. Grouping
1)E2And (4) screening and grouping the safety concentration:
blank group: after culturing for 24h by using the DMEM culture solution, replacing the DMEM culture solution once, and continuously culturing for 24 h;
E2group (2): after 24h of DMEM culture solution culture, the final concentration is changed to 10 mu mol/L, 1 mu mol/L and 10 mu mol/L respectively-1μmol/L、10-2μmol/L、10-3μmol/L、10-4μmol/L、10-5Mu mol/L of E2Continuously culturing the solution for 24 hours;
2)E2screening and grouping effective concentration:
blank group: after culturing for 24h by using the DMEM culture solution, replacing the DMEM culture solution once, and continuously culturing for 26 h;
model group: culturing in DMEM culture solution for 24 hr, replacing DMEM culture solution, culturing for 2 hr, and administering A beta25-35 solution, with a final concentration of 20 μmol/L, and further culturing for 24 h;
E2+Aβ25-35group (2): culturing in DMEM culture solution for 24 hr, and changing to final concentrations of 1 μmol/L and 10 μmol/L-1μmol/L、10-2μmol/L、10-3μmol/L、10-4μmol/L、10-5Mu mol/L of E2Incubating for 2h, and administering A beta25-35The solution was allowed to stand at a final concentration of 20. mu. mol/L and the culture was continued for 24 hours
3. MTT assay
1) The cell suspension in log phase was seeded into 96-well plates at 2500 cells/200 μ Ι _ per well, with 6 replicates per group;
2) adding 20 mu L/hole of MTT solution (5g/L), and continuing to incubate for 4h at 37 ℃;
3) discarding the waste liquid, adding DMSO (150 μ L/hole), and oscillating on a constant temperature oscillator for 10 min;
4) the absorbance (OD) of each well was measured at 570nm with a microplate reader.
4. Statistical treatment
The results were processed with SPSS18.0 software to
Showing that the comparison between groups is processed by using one-way variance, the comparison between two samples is tested by LSD, P<The difference was significant at 0.05.
5. Results of the experiment
1)E2The results of screening for safe concentrations are shown in Table 1, with E at 10. mu. mol/L compared to the blank2The proliferation rate of the group cells is obviously reduced (P)<0.01)，1、10-1、10-2、10-3、10-4And 10-5Mu mol/L of E2The proliferation rate of the group cells has no obvious change. It can be seen that the safe concentration range of E2 is 1, 10-1、10-2、10-3、10-4And 10-5Mu mol/L, can be used for the subsequent screening of the effective concentration of E2.
TABLE 1E2Effect on the proliferation Rate of Normal PC12 cells (
n＝6)
Note: p <0.01 as compared to blank
2)E2The results of screening for effective concentration are shown in Table 2, and the cell proliferation rate in the model group is significantly decreased (P) as compared with that in the blank group<0.01); comparison with model groups, 1, 10-1、10-2、10-3、10-4And 10-5Mu mol/L of E2The proliferation rate of the group cells is obviously increased (P)<0.01，P<0.05), wherein, 10-3Mu mol/L of E2The group cells had the highest proliferation rate, so E was selected210 of-3The concentration of mu mol/L is the optimal effective concentration for subsequent experimental study.
TABLE 2E2For Abeta25-35Effect of impaired proliferation Rate of PC12 cells (
n＝6)
Note: is P compared with blank group<0.01, compared to the model set,##is P<0.01，#Is P<0.05。
Example 2 detection of Oriental cherry blossom glycoside vs. Abeta by MTT method25-35Effect on the viability of PC-12 cells
1. Cell culture procedure as in example 1
2. Grouping
1) The concentration screening of the sakura-like glycosides is divided into groups:
blank group: after culturing for 24h by using the DMEM culture solution, replacing the DMEM culture solution once, and continuously culturing for 24 h;
sakura group: after 24h of DMEM culture solution culture, the final concentration is changed to 4 multiplied by 10 respectively2μmol/L、40μmol/L、4μmol/L、4×10-1μmol/L、4×10-2μmol/L、4×10-3μmol/L、4×10-4μmol/L、4×10-5Continuously culturing the micro mol/L of the primrose-hip glycoside solution for 24 h;
2) screening and grouping the effective concentration of the rosa laevigata:
blank group: after culturing for 24h by using the DMEM culture solution, replacing the DMEM culture solution once, and continuously culturing for 26 h;
model group: culturing in DMEM culture solution for 24 hr, replacing DMEM culture solution, culturing for 2 hr, and administering A beta25-35The solution is cultured for 24 hours continuously, and the final concentration of the solution is 20 mu mol/L;
oriental cherry blossom glycoside + Abeta25-35Group (2): culturing in DMEM culture solution for 24 hr, and changing to final concentration of 4 × 10-1μmol/L、4×10-2μmol/L、4×10-3μmol/L、4×10-4μmol/L、4×10-5Mu mol/L of sakura glycoside solution is cultured for 2h and then A beta is given25-35The solution is cultured for 24 hours continuously, and the final concentration of the solution is 20 mu mol/L;
3. MTT assay procedure as in example 1
4. Statistical treatment
The results were processed with SPSS18.0 software to
Showing that the comparison between groups is processed by using one-way variance, the comparison between two samples is tested by LSD, P<The difference was significant at 0.05.
5. Results
1) The results of screening for the safe concentration of sakuranetin are shown in Table 3, which is 4X 10 in comparison with the blank group2The proliferation rate of the primeveroside group cells of mu mol/L is obviously reduced (P)<0.01), the proliferation rate of 40 mu mol/L and 4 mu mol/L of Jiangfu cherry blossom bud group cells is obviously increased (P)<0.01)，4×10-1μmol/L、4×10-2μmol/L、4×10-3μmol/L、4×10-4μ mol/L and 4X 10-5Mu mol/L of the sakura roseoside group cell proliferation rate has no obvious change. It is known as 4X 10-1μmol/L、4×10-2μmol/L、4×10-3μmol/L、4×10-4μ mol/L and 4X 10-5Mu mol/L of sakura has no obvious influence on cell proliferation. Therefore, the safe concentration range of the sakura-like glycosides is 4 multiplied by 10-1μmol/L、4×10-2μmol/L、4×10-3μmol/L、4×10-4μ mol/L and 4X 10-5Mu mol/L, can be used for the subsequent screening of the effective concentration of the sakura.
TABLE 3 Effect of Oriental cherry blossom glycosides on the proliferation Rate of Normal PC12 cells (
n＝6)
Note: p <0.01 as compared to blank
2) The results of screening the effective concentration of sakura are shown in Table 4, and the cell proliferation rate of the model group is significantly reduced (P) compared with that of the blank group<0.01); compared with model group, 4 × 10-1The proliferation rate of the micro mol/L Jianghua flowering cherry group cells is obviously increased (P)<0.01)，4×10-2μmol/L、4×10-3μmol/L、4×10-4μ mol/L and 4X 10-5Mu mol/L of the sakura roseoside group cell proliferation rate has no obvious change. The effective concentration of sakura-like glycosides is 4 × 10-1Mu mol/L, can be used for subsequent experimental study.
TABLE 4 Oriental cherry blossom glycoside vs. Abeta25-35Effect of damaging the Activity of PC-12 cells: (
n＝6)
Note: is P compared with blank group<0.01, compared to the model set,##is P<0.01。
Example 3 Oriental cherry glycoside vs. Abeta25-35Study of protective Effect of induced PC12 cell injury
1. Cell culture procedure as in example 1
2. Grouping
Blank group: after culturing for 24h by using the DMEM culture solution, replacing the DMEM culture solution once, and continuously culturing for 26 h;
model group: culturing in DMEM culture solution for 24 hr, replacing DMEM culture solution, culturing for 2 hr, and administering A beta25-35The solution is cultured for 24 hours continuously, and the final concentration of the solution is 20 mu mol/L;
E2+Aβ25-35group (2): culturing in DMEM culture solution for 24 hr, and changing to final concentration of 10-3Mu mol/L of E2Incubating for 2h, and administering A beta25-35The solution is cultured for 24 hours continuously, and the final concentration of the solution is 20 mu mol/L;
oriental cherry blossom glycoside + Abeta25-35Group (2): culturing in DMEM culture solution for 24 hr, and changing to 4 × 10-1Mu mol/L of sakura glycoside solution is cultured for 2h, and A beta is given25-35The solution was allowed to stand at a final concentration of 20. mu. mol/L, and the culture was continued for 24 hours.
3. MTT assay procedure was as in example 1
4. Statistical treatment
The results were processed with SPSS18.0 software to
Showing that the comparison between groups is processed by using one-way variance, the comparison between two samples is tested by LSD, P<The difference was significant at 0.05.
5. Results
As a result, as shown in Table 5, the cell proliferation rate was significantly decreased in the model group (P) as compared with that in the blank group<0.01); compared with the model group, the sakura glycosides + Abeta25-35The proliferation rate of the group cells is obviously increased (P)<0.01). The fact that sakura glycosides of Jianghu province are to Abeta25-35The induced PC12 cell activity injury has the protection function and the effect2Similarly.
TABLE 5 Oriental cherry blossom glycoside vs. Abeta25-35Effect of damaging the Activity of PC-12 cells: (
n＝6)
Note: is P compared with blank group<0.01, compared to the model set,##is P<0.01。
Example 4 Oriental cherry glycoside vs. Abeta25-35Effect of induced apoptosis of PC12 cells
1. The cell culture procedure was as in example 1
2. Grouping same as example 3
3. Flow cytometry for detecting apoptosis rate of cells
1) Cell suspensions in log phase were seeded into 6-well plates, 4 × 105Culturing each cell at 2 ml/hole;
2) the supernatant was pipetted into a 15ml centrifuge tube, the cells in the 6-well plate were washed once with PBS, 0.25% trypsin was added, and then digestion was stopped with the aspirated supernatant;
3) the cells were harvested by centrifugation, 1ml of PBS was added to resuspend the cells and counted, taking the cell containing about 105Resuspension of individual cells, centrifugation at 1000rpm for 5min and removal of supernatant;
4) and (4) carrying out flow cytometry detection according to the operation of the apoptosis kit.
4. Statistical treatment
The results were processed with SPSS18.0 software to
Showing that the comparison between groups is processed by using one-way variance, the comparison between two samples is tested by LSD, P<The difference was significant at 0.05.
5. Results
As shown in Table 6, the apoptosis rate was significantly increased in the model group (P) as compared with that in the blank group<0.01); compared with the model group, the sakura glycosides + Abeta25-35The apoptosis rate of the group cells is obviously reduced (P)<0.01). This indicates that sakura glycosides can inhibit A beta25-35Induced apoptosis, effects and E2Similarly.
TABLE 6 Oriental cherry blossom glycoside vs. Abeta25-35Effect of impaired apoptosis of PC-12 cells (
n＝6)
Note: is P compared with blank group<0.01, compared to the model set,##is P<0.01。
Example 5 Oriental cherry glycoside vs. Abeta25-35Influence of ROS, MDA and SOD content change in damaged PC12 cells
1. The cell culture procedure was as in example 1
2. Grouping same as example 3
3. Determination of T-SOD
1) Cell suspensions in log phase were seeded into 6-well plates, 4 × 105Culturing each cell at 2 ml/hole;
2) sucking the supernatant into 15ml centrifuge tubes, adding RIPA lysate (containing 1% PMSF) into each centrifuge tube, standing for 3min, transferring all the liquid into 1.5ml centrifuge tubes, and standing for 30 min;
3) centrifuging at 4 deg.C and 12000rpm for 5min, and collecting supernatant in 1.5ml centrifuge tube;
4) protein quantification is carried out by using a BCA kit, and OD values of each group are determined by using a semi-automatic biochemical analyzer at the wavelength of 550nm according to the steps of the total superoxide dismutase test kit specification. The total SOD value in each group of cells was calculated using the following formula:
4. determination of MDA
1) The extraction steps of the protein are the same as 1) -3 in the T-SOD determination);
2) each set of OD values was determined at 532nm wavelength using a semi-automatic biochemical analyzer according to the procedure of the malondialdehyde test kit instructions. The MDA content in each group of cells was calculated using the following formula:
5. ROS assay
1) Cell suspensions in log phase were seeded into 6-well plates, 4 × 105Culturing each cell at 2 ml/hole;
2) removing the culture solution, adding 10 mu mol/L DCFH-DA 1 ml/hole, placing the 6-hole plate in an incubator, standing for 20min, and mixing the holes in the hole plate once every 5min to make the cells fully contact with the probe;
3) and washing the cells for three times by using the serum-free culture solution, collecting the cells, adding 1ml of the serum-free culture solution, and detecting by using an up-flow cytometer.
6. Statistical treatment
The results were processed with SPSS18.0 software to
Showing that the comparison between groups is processed by using one-way variance, the comparison between two samples is tested by LSD, P<The difference was significant at 0.05.
7. Results
As shown in Table 7, the contents of ROS and MDA in the model cells were significantly increased and the SOD activity was significantly decreased (P) as compared with that in the blank group<0.01); compared with the model group, the sakura glycosides + Abeta25-35The content of ROS and MDA in the histiocyte is obviously reduced, and the SOD activity is obviously improved (P)<0.01). Shows that the primrose glycoside can protect Abeta25-35Induced oxidative damage of PC12 cells, its role in connection with E2Similarly.
TABLE 7 Abeta25-35(expression of damaged ROS, MDA and SOD content in PC12 cell: (
n＝6)
Note: p is compared with blank group<0.01; in comparison to the model set,##is P<0.01。
Example 6 Oriental cherry glycoside vs. Abeta25-35Effect of impaired p-Tau protein expression in PC12 cells
1. The cell culture procedure was as in example 1
2. Grouping into specific groups as in example 3
3. Western Blot for detecting protein content
1) Extraction of Total cellular protein
Collecting each group of cells into a 15ml centrifuge tube, washing twice with 4 ℃ precooled PBS, adding 300 mul of RIPA lysate (containing 1% PMSF) into each culture bottle, standing for 3min, transferring the liquid into a 1.5ml centrifuge tube, standing for 30min, then centrifuging (4 ℃, 12000rpm, 5min), and sucking out the supernatant into another new 1.5ml centrifuge tube;
2) denaturation of proteins
Protein samples were mixed with 5 x SDS loading buffer at 4: mixing at a ratio of 1, heating in boiling water for 7min to denature protein;
3) SDS-PAGE electrophoresis
Preparing 10% separation gel and 5% concentrated gel, adding 60 μ g of denatured protein sample, and performing electrophoresis under conditions of concentrated gel 80V and separation gel 100V;
4) rotary film
(1) Preparing a PVDF membrane and two pieces of same filter paper, sequentially placing the PVDF membrane and the two pieces of same filter paper in methanol, deionized water and a membrane transferring buffer solution for soaking, paving the filter paper, the PVDF membrane, the gel and the filter paper in sequence from bottom to top, removing bubbles from a glass plate, placing the glass plate on a semi-dry transfer printing instrument, and keeping the temperature for 10V for 30 min;
5) sealing of
Taking out the PVDF membrane, and incubating for 2h in a prepared sealing solution at room temperature;
6) antibody incubation
Taking out the PVDF membrane, placing the PVDF membrane in a hybridization bag, reserving a gap, adding about 1ml of primary anti-dilution solution to enable the front surface of the PVDF membrane to be fully contacted with the PVDF membrane, sealing the PVDF membrane, and standing overnight at 4 ℃; pouring out the primary anti-dilution solution, washing for three times by TBST, and washing for 5 min/time; taking out the PVDF membrane, placing in a hybridization bag, adding about 1ml of prepared second antibody diluent into the bag, sealing, and standing at room temperature for 1 h;
7) washing membrane
Pouring out the secondary antibody diluent, washing for three times by TBST (tert-butyl ether) for 5 min/time;
8) chemiluminescence
Mixing ECL luminous liquid A and B in the same amount, dripping onto PVDF film to cover it uniformly, exposing and developing in dark room, and analyzing
4. Statistical treatment
The results were processed with SPSS18.0 software to
Showing that the comparison between groups is processed by using one-way variance, the comparison between two samples is tested by LSD, P<The difference was significant at 0.05.
5. Results
As shown in Table 8, the phosphorylation levels of Tau protein in the cells of the model group were significantly increased as compared with the blank group (P)<0.01), sakuranetin + Abeta compared with model group25-35The phosphorylation level of the group Tau protein is obviously reduced (P)<0.01), action with E2Similarly. The fact that the rosa laevigata glucoside can inhibit A beta toxicity by inhibiting Tau protein phosphorylation level and play a role in protecting cells is suggested.
TABLE 8 Abeta25-35Expression of p-Tau protein in injured PC12 cells: (
n＝6)
Note: blank group comparison, with P<0.01, model group comparison,##is P<0.01。
Example 7 detection of ER β, p-Akt, t-Akt, p-GSK-3 β, t-GSK-3 β, p-Tau, t-Tau and caspase-3 in PC-12 cells by Western blot
In order to study the effect of sakura on proteins in PC12 cells, ER antagonist ICI182,780 was used for intervention to observe changes in protein molecules in sakura-treated cells.
1. The cell culture procedure was as in example 1
2. Grouping:
blank group (1), model group (2), E2+Aβ25-35Group (3), Jianghua cherry blossom glycoside + Abeta25-35Group (4) same as example 3;
E2+Aβ25-35+ ICI182,780 set (5): culturing in DMEM culture solution for 24h, replacing with ICI182 with final concentration of 1 μmol/L, culturing for 1h, replacing with ICI with final concentration of 10-3Mu mol/L of E2Incubating for 2h, and administering A beta25-35The solution is cultured for 24 hours continuously, and the final concentration of the solution is 20 mu mol/L;
oriental cherry blossom glycoside + Abeta25-35+ ICI182,780 set (6): culturing in DMEM culture medium for 24h, replacing with ICI182 with final concentration of 1 μmol/L, culturing for 1h in 780, and replacing with final concentration of 4 × 10-1Mu mol/L of sakura glycoside solution is cultured for 2h, and A beta is given25-35Solution of, makingThe final concentration is 20 mu mol/L, and the culture is continued for 24h
3. The specific steps for detecting the expression of ER beta, p-Akt, t-Akt, p-GSK-3 beta, t-GSK-3 beta, p-Tau, t-Tau and caspase-3 protein by Western blot method are the same as example 6
4. Statistical treatment
The results were processed with SPSS18.0 software to
Showing that the comparison between groups is processed by using one-way variance, the comparison between two samples is tested by LSD, P<The difference was significant at 0.05.
5. Results
As a result, as shown in Table 9, the expression of ER β, P-Akt/t-Akt and P-GSK-3 β/t-GSK-3 β in the cells of the model group was significantly reduced (P) as compared with that of the blank group<0.01), the content of P-Tau/t-Tau and caspase-3 is obviously increased (P)<0.01); compared with the model group, the sakura glycosides + Abeta25-35The expression of ER beta, P-Akt/t-Akt and P-GSK-3 beta/t-GSK-3 beta in the cells is obviously increased (P)<0.01), the expression of P-Tau/t-Tau and caspase-3 was significantly reduced (P)<0.01); with sakura glycoside + Abeta25-35Group comparison, Jiangfu Yinghua glycoside + Abeta25-35+ ICI182,780 group cells with significantly reduced expression of ER β, P-Akt/t-Akt and P-GSK-3 β/t-GSK-3 β (P)<0.01), the content of P-Tau/t-Tau and caspase-3 is obviously increased (P)<0.01). Action effect of Jiangfu Yinghua glycoside and E2Similarly. The presence of ER antagonist can reduce the A beta pair of sakura25-35The inhibitory action of (1). Shows that the sakura glycoside can activate Akt through activating ER pathway and activate GSK-3 beta so as to reduce hyperphosphorylation level of Tau protein and inhibit apoptosis, and can treat A beta25-35Induced PC12 cell damage exerts a protective effect.
TABLE 9 ER β, p-Akt/t-Akt, p-GSK-3 β/t-GSK-3 β, p-Tau/t-Tau and caspase-3 proteins in PC-12 cells: (
n＝3)
Note: p is compared with blank group<0.01; in comparison to the model set,##is P<0.01; compared with the administration group, the composition has the advantages that,++is P<0.01
Example 8 combination of drug pairs for Abeta25-35Study of protective Effect of induced PC12 cell injury
To evaluate different combinations of phytohormones on Abeta25-35Induced PC12 cell damage protection effect, the inventors performed experiments using the checkerboard method.
1. Cell grouping and culturing
Cell grouping and culture As in example 1, the drug combination was incubated for 2 hours with two test drugs diluted at different fold ratios and then administered with A.beta.25-35The solution was allowed to stand at a final concentration of 20. mu. mol/L, and the culture was continued for 24 hours.
2. MTT assay procedure was as in example 1
3. Data statistics
The results of the experiment were analyzed using MacSynergyII software.
4. Results
The composition is prepared by combining Orientin, luteolin-7-O-beta-D neohesperidin, naringin, Orientin-7-O-beta-D-glucoside, luteolin-7-O-beta-D-glucoside, neoeriocitrin, eriodictyol, North America eriodictyol and kaempferol in different ways to protect A beta25-35The induced PC12 cell injury has different effects such as synergy and superposition, and the results are shown in Table 10, wherein the sakuranetin and luteolin-7-O-beta-D neohesperidin, eriodictyol or kaempferol have strong synergistic effect.
TABLE 10 combination pair Abeta25-35Induced protection of PC12 cell injury
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Claims (5)
1. A pharmaceutical composition for treating Alzheimer disease is selected from an effective amount of the effective component of rosaceous cherry blossom glycoside; and any one of luteolin-7-O-beta-D neohesperidin, naringenin-7-O-beta-D-glucoside, luteolin-7-O-beta-D-glucoside, eriodictyol and kaempferol.
2. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier or excipient.
3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is in the form of a powder, a gel or a liquid.
4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is in the form of a tablet, a wafer capsule, a gel capsule, a stick, a sachet, a vial, a dropper, or an injectable form.
5. Use of a pharmaceutical composition according to any one of claims 1 to 4 for the manufacture of a product for the treatment of alzheimer's disease.
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| CN101156883A (en) * | 2007-10-24 | 2008-04-09 | 石任兵 | Stevia rebaudian valid target as well as its activity and application |
| CN101292990A (en) * | 2008-06-25 | 2008-10-29 | 通化华夏药业有限责任公司 | Use of luteolin-7-O-beta-D glucoside in preparing medicaments for treating dementia diseases |
| CN107660124A (en) * | 2015-06-03 | 2018-02-02 | Mjn 美国控股有限责任公司 | Alimentation composition and its application containing elevated horizontal inositol |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101156883A (en) * | 2007-10-24 | 2008-04-09 | 石任兵 | Stevia rebaudian valid target as well as its activity and application |
| CN101292990A (en) * | 2008-06-25 | 2008-10-29 | 通化华夏药业有限责任公司 | Use of luteolin-7-O-beta-D glucoside in preparing medicaments for treating dementia diseases |
| CN107660124A (en) * | 2015-06-03 | 2018-02-02 | Mjn 美国控股有限责任公司 | Alimentation composition and its application containing elevated horizontal inositol |
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